1. Field of the Invention
The present invention relates generally to apparatus and methods for automatically isolating preparative quantities of a substance from a mixture of substances in a sample liquid by sequentially fractionating the sample and contacting the fractions with reagent liquids by means of a centrifugal rotor containing at least one fractionation cell and a dynamic flow distributor which, in combination, allow reagent liquids to flow into and sequentially through one or more compartments of the fractionation cell while the rotor is rotating.
2. Description of the Background Art
Many manual methods involving sedimentation, aggregation, phase separation, sorption, rinsing, desorption and other manipulations for isolating a substance from a sample liquid are well known. Although several of these procedures include centrifugation steps, manual interventions such as pipetting, pouring, mixing, shaking, decanting, etc. are typically required between individual steps.
In a preferred embodiment of the present invention, one or more nucleic acids are isolated from a sample liquid. Typically, the sample liquid initially contains a mixture of biological materials such as cells, dissolved proteins, and nucleoprotein complexes. Manual methods for isolating nucleic acids contained in such mixtures typically begin with lysis, protein denaturation or other manipulations to release elements that contain the nucleic acids sought to be isolated. Residual formed elements are then either trapped by filtration or else centrifuged into a pellet. The cleared sample liquid is then typically contacted with a reagent liquid which neutralizes or otherwise conditions the sample liquid and, optionally, conditions the nucleic acids sought to be isolated. Subsequent steps may include contacting the conditioned sample liquid with one or more reagents which further condition the sample liquid. Optionally, the sample liquid, with or without further conditioning, may contact a material such as a gel, resin, membrane, glass or other surface which selectively retains the nucleic acid sought to be isolated, or a derivative thereof, by adsorption or absorption. Other steps include removing substances with which the nucleic acid may be complexed and extracting the nucleic acid from solution or from the adsorptive or absorptive material.
Attempts to automate the aforesaid manual procedures have heretofore relied generally upon robotic means. The AUTOGEN 540, manufactured by Autogen Instruments, Inc., is an example. Another is the GENEPURE 341 manufactured by Applied Biosystems, Inc. Centrifugation is only incidental to the operation of the former and is not involved in the latter.
Columbus, et al. (U.S. Pat. No. 5,032,288) have disclosed a centrifugal method for extracting deoxyribonucleic acid by phase separation. To our knowledge, however, centrifugal means have not been adapted to carry out multi-step chemical procedures for isolating either substances in general or nucleic acids in particular within the confines of a rotor into which reagents are introduced as the rotor rotates. Such means, which lend themselves readily to automation, are likely to be faster, more accurate and reproducible, and less labor-intensive than manual methods. They are likely to be less expensive to manufacture and maintain than robotic means.
In contrast to their limited employment in multi-step chemical preparative procedures, centrifugal rotors for chemical analyses are common. Typically, they have been variously adapted for separating blood plasma from whole blood and for performing chemical analyses on the separated plasma.
Burd (U.S. Pat. No. 5,061,381) describes a centrifugal rotor which separates plasma from whole blood and, by means of a plurality of internal chambers and passages, combines the plasma with one or more reagents and distributes it to a plurality of individual test wells. In the Burd rotor and other disclosed analytical rotors (e.g., U.S. Pat. Nos. 4,756,883, 4,663,296, 4,244,916), and in the instant invention, a capillary limits the flow of liquid between chambers when the rotor is static. Centrifugal force is required to urge liquid through the capillary. Other analytical rotors similarly employ a plurality of chambers to separate plasma into aliquots, treat it, and mix it with reagents (exemplarily, Anderson U.S. Pat. No. 3,586,484). Also conventional is the use of a combination of centrifugal and gravitational force to control the movement of plasma and reagents between chambers. Additionally, the direction of rotation may be exploited for this purpose (e.g., Guigan, U.S. Pat. Nos. 4,463,097 and 4,469,793).
Typically, rotors adapted for analyzing blood plasma include centrifugal means for distributing substantially equal aliquots of a plasma sample to a plurality of test chambers. Unlike the present invention, however, the distributor means is usually loaded while the rotor is static. This limitation can result in uneven distribution of sample aliquots. A variety of venting systems and overflow chambers have been disclosed to deal with this difficulty. See, for example, Burtis, et al., U.S. Pat. No. 3,901,658. Dynamic loading of liquids has been employed in an effort to overcome the problem of uneven distribution. Anderson, et al. U.S. Pat. No. 3,873,217, for example, have disclosed an analytic rotor capable of being dynamically loaded. However, any unevenness of flow during the injection of liquids into dynamic flow distributors has generally resulted in eneven distribution of sample aliquots.
The aim of the invention is to provide easily automated, centrifugal apparatus and methods for the preparative isolation of a substance from a mixture of substances in a sample liquid, in particular, a nucleic acid. Advantages of such apparatus and methods over the current art include better productivity, accuracy and reproducibility than manual methods, lower manufacturing and maintenance costs than robotic systems, and greater flexibility than other centrifugal means now used for preparative purposes or adaptable therefor.